Confocal mosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology

Abstract

Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12 × 12 mm (which is equivalent to a 2×-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

“…Rapid micrographic pathology represents an opportunity for confocal microscopy of whole tissue excisions. By stitching square raster-scanned fields of view together, a 12 × 12 mm mosaic can be produced in 9 min, 12 which is similar to a 2× histological view of a Mohs excision. A recently developed "strip mosaicing" technique is even faster, producing a 10 × 10 mm mosaic in about 3 min.…”

“…We present an important step in a series of advances in confocal mosaic microscopy as an augmentation or alternative to frozen histology the use of eosin (Eo) in fluorescence mode to identify cytoplasm in confocal mosaics. Thus far, only two contrast mechanisms have been investigated for (1) nuclei in fluorescence mode with acridine orange 12 (AO), and (2) collagen and keratin in endogenous reflectance (R) mode. 15 The combination of these two modes works to detect BCC because the nuclear stain provides a clear positive indicator of the dense nuclear morphology on the background of counter contrasted collagen and keratin.…”

“…These are rapid techniques compared to histology, which takes 20 to 45 min. 12 Adapting the technique to guide excision of different cancer types would save more tissue and possibly enable new surgeries. Expanding within the framework of Mohs surgery, the pathologic profile of squamous cell carcinoma may be applicable beyond the skin.…”

A Viterbi Approach to Topology Inference for Large Scale Endomicroscopy Video Mosaicing

“…In the second category are methods adapted for known -usually robotizedacquisition trajectories (e.g. microscope with motorized platform) where the a priori knowledge directly provides the topology [3]. For our project, a dedicated device was designed to allow video mosaic acquisitions wherein a robot holds the probe and follows a pre-defined spiral trajectory [2].…”

Scanning in situ Spectroscopy platform for imaging surgical breast tissue specimens

“…But the conventional microscopic confocal systems [14] are optimized for high resolution, depthsectioned imaging over narrow fields of view, which again is not suitable for imaging cancer margins across large fields. While mosaic imaging techniques could be used to extend the field of view [15,16], these designs do not maintain a consistent sampling geometry at each pixel position, which makes it particularly unsuitable for imaging tissue scatter signals that are known to exhibit spatial and angular dependence [17].…”